Optimization of the flash extraction of flavonoids from ...42 Introduction 43 Currently, there are increasing applications of flavonoids in food and medicine for their 44 extensive

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3 Optimization of the flash extraction of flavonoids from the leaves of Salix babylonica using the

4 response surface method and an evaluation of the leaves’ high antioxidant activity

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6 Huijie Chen1, 2, Lei Diao2, Yue Zhang1, Haixin Liu1, Ming Zhong1, Guangxing Li1*

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8 1 Department of Pharmaceutical Preparation, Jilin Agriculture Science and Technology University,

9 Jilin, China

10 2 Department of Basic Veterinary, Northeast Agricultural University, Harbin, China

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12 *Corresponding author

13 Email: [email protected] (G X L)

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.CC-BY 4.0 International licenseavailable under awas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made

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24 Abstract25 Many biological activities of Salix babylonica leaves are attributed to the plants’ high total

26 flavonoid content. Flash extraction has the advantages of high efficiency and maximum retention of

27 the active ingredient. In this study, flash extraction was used to extract the total flavonoids, and a

28 Box–Behnken design was used to optimize the extraction conditions for the first time. The effects

29 of four independent variables, including ethanol concentration, extraction voltage, time, and ratio of

30 liquid to material on flavonoid yield, was determined, and the optimal conditions for flavonoid

31 extraction were evaluated using response surface methodology. Statistical analyses showed that the

32 linear and quadratic terms of these four variables had significant effects. The fitted second-order

33 model revealed that the optimal conditions consisted of an ethanol concentration of 67.91%,

34 extraction time of 87 s, extraction voltage of 116 V and ratio of liquid to material of 42.79. Under

35 the optimum conditions, the experimental value of 66.40±0.80% nearly coincided with that

36 predicted by the model. In the ferric reducing antioxidant power (FRAP) and

37 2,2-diphenyl-1-picrylhydrazyl radical (DPPH.) assays, the extracts showed significant antioxidant

38 and scavenging capacity for free radicals, respectively. This study helps to better exploit the

39 resources of Salix babylonica leaves and provides new insights for effective extraction of

40 flavonoids.

41

42 Introduction43 Currently, there are increasing applications of flavonoids in food and medicine for their

44 extensive biological functions in terms of antibacterial, antiviral and antioxidant activities [1-5]. As

45 a biological response modifier, most of these flavonoids are derived from traditional Chinese

46 medicine, including Salix babylonica. This plant is economically notable and is distributed



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47 throughout Asia, Europe, and the Americas. The leaf of Salix babylonica has been studied in China

48 for antifungal and anti-obesity utilization due to its high content of flavonoids [6, 7]. Currently,

49 there are limited studies on the extraction process and antioxidant activity of the total flavonoids

50 from Salix babylonica leaves [8], and the primary focus is on the enrichment and purification of the

51 total flavonoids. In addition, most of the extraction methods continue using the outmoded methods

52 of reflux extraction, ultrasonic extraction and microwave extraction [9, 10]. In these cases,

53 extraction required a long time and was labor- and material-intensive, and the most important factor

54 was that the active ingredients of the extract were easily degraded. To use Salix babylonica leaf

55 resources more optimally, there is an urgent need to establish the optimum procedure for the

56 effective extraction of flavonoids.

57

58 The flash extraction method is a new method in recent years [11, 12]. This method uses the

59 principle of high-speed shear force and molecular filtration to break up the stems, leaves, roots and

60 other materials of plants into tiny particles such that the concentration of the extraction solvent

61 reaches equilibrium in a short time [13, 14]. In addition, the solute transfer process is completed in

62 tens of seconds or even seconds, and the active ingredients in the plant are only destroyed to a small

63 extent. Compared with the traditional hot water extraction, enzyme extraction and other physical

64 field-assisted extraction methods, such as microwave, ultrasonic, and ultrahigh pressure extraction,

65 the flash extraction method uses small amounts of solvent, a short extraction time and is highly

66 efficient [15-18]. In recent years, there have been many reports of the use of this technology to

67 extract phytochemicals. It was used to extract water-soluble components, such as polyphenols [19],

68 glycosides [20], and polysaccharides [21], as well as water-insoluble components, such as tannins

69 [22], peony [23], and volatile oil [24]. Therefore, the flash extraction method with its unique



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70 advantages of speed, energy, and the savings in solvents is bound to exert greater potential for

71 herbal extractions.

72

73 In humans, oxidation is involved in the generation of energy, but the body may be affected by

74 a variety of factors, such as illness, aging, and free oxygen radicals produced by oxidation, that are

75 uncontrollable and harmful to the human body. Antioxidants, such as flavonoids, can eliminate

76 these free radicals, reduce the risk of death from related diseases, and slow aging [25, 26].

77 Therefore, the preparation and application of antioxidants to protect the body from free radical

78 damage is popular [27]. In this study, flash extraction and response surface methodology (RSM)

79 were combined to seek an effective extraction procedure. In addition, the antioxidant capacity of the

80 extracts was investigated to verify the effectiveness of flash extraction. The results indicated that

81 the flash extraction method could maximize the protection of the active ingredients of flavonoids

82 with high biological function. These findings will provide a theoretical basis to develop and utilize

83 Salix babylonica leaf resources.

84

85 Materials and methods

86 Materials

87 The leaves of Salix babylonica were collected in Jilin Province (China). Rutin and oligomeric

88 proanthocyanidins (OPC) standards were purchased from the National Institutes for Food and Drug

89 Control (Beijing, China). Aluminum nitrate, sodium hydroxide, ethanol, sodium nitrite, hexahydrate

90 ferric chloride and ferrous sulfate were analytically pure and obtained from the Tianjin Reagent

91 Company (Tianjin, China). 2,4,6-tri(2-pyridyl)-1,3,5-triazine (TPTZ) and

92 2,2-diphenyl-1-picrylhydrazyl radical (DPPH) were purchased from J&K Scientific Co. (Beijing,



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93 China).

94

95 Extraction of total flavonoids

96 The leaves of Salix babylonica were crushed using a grinder and passed through a 20-mesh

97 screen. With the use of a flash extractor, a number of samples were accurately weighed and

98 extracted under different extraction times, extraction numbers, ratio of liquid to raw material,

99 ethanol concentration and extraction voltage. The extract was separated from the insoluble residue

100 using centrifugation (3000 rpm, 5 min), and the extract was filtered using a 0.45 μm microporous

101 membrane. The filtrate was concentrated and dissolved to 100 mL to determine the concentration of

102 the flavonoids.

103

104 Determination of the flavonoid contents

105 The colorimetric method was used to determine the content of the flavonoids in the extract [28,

106 29]. One milliliter of flavonoid extract, 1 mL of 5% (w/w) sodium nitrite and 5 mL of 80% (v/v)

107 ethanol were accurately measured and mixed for 6 min. One milliliter of 10% (w/w) aluminum

108 trichloride were added and incubated for 6 min, and 10 mL of 1 mol/L sodium hydroxide was added.

109 The absorbance of the solution was measured using a UV-2900 spectrophotometer at 510 nm after

110 15 min. A blank control was used. The standard curve (y = 0.0164 + 0.0097x, where y is absorbance

111 value of sample, and x is sample concentration) ranged from 16.32–65.28 μg/mL (R2=0.9993).

112

113 Experimental design

114 The extraction rate of the flavonoids is influenced by many factors， such as the extraction

115 voltage, extraction time, the ratio of liquid to raw material, ethanol concentration and extraction



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116 number [30, 31]. Based on the single factor test results, factors that have a significant impact on the

117 flavonoids were selected to test using response surface methodology. The four significant impact

118 factors were designated X1, X2, X3, and X4, and each factor was set at three levels, coded +1, 0, −1

119 for high, medium and low values, respectively (Table 1). Four variables were encoded based on the

120 following equation:

121 0ii

x xXx

， 1 4i (1)

122 where Xi represented the encoded value of the variable; xi represented the actual value of the

123 variable; x0 represented the actual value of the independent variable at the center point, and x

124 represented the change value of the variable, respectively.

125

126 Table 1. Variables and design levels of response surface methodology.

Independent variables Coded symbolsLevels

-1 0 1

Extraction voltage (V) X1 110 120 130

Ethanol concentration (%) X2 50 60 70

Extraction time (s) X3 80 90 100

Ratio of liquid to material X4 30 40 50

127

128 To use the Box-Behnken design and obtain the best extraction conditions, a second order

129 polynomial model was used to illustrate the relation between the response values and independent

130 variables. The model is as follows:

1314 4 4 4

20

0 0 0 0i i ii i ij i j

i j i jY X X X X

(2)

132 Where Y represented the response function; β0 represented the constant, and βi, βii and βij



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133 represented the coefficients of the linear, quadratic and interactive terms, respectively. Xi, Xi2 and

134 XiXj were the encoded independent variables, interaction and quadratic terms, respectively.

135

136 To design the experiments and analyze the experimental data, design-expert software (version

137 8.0) was used. Based on the analysis of variance, the regression coefficients of individual linear,

138 quadratic, and interaction terms were determined. The regression coefficients were used to provide

139 statistical calculations to generate dimensional and contour maps from the regression model. The

140 lack of fit and the coefficient of determination (R2) were generated using the Design-Expert

141 software. They represented the accuracy of the model. Fischer’s F-test at a probability (P) of 0.001,

142 0.01 or 0.05 examined the significance of the model, as well as the encoded independent variables,

143 interaction and quadratic terms.

144

145 Antioxidant activity assay of flavonoids

146 A ferric reducing antioxidant power (FRAP) experiment was used to measure the total

147 antioxidant activity of the extracts and standards (FeSO4 solution). A 2,2-diphenyl-1-picrylhydrazyl

148 radical (DPPH) experiment was used to measure the free radical scavenging activity of the extracts

149 and standards using OPC.

150

151 FRAP assay

152 FRAP reagents included 50 mmol/L acetate buffer, which contained 20.4 g C2H3NaO2 and 80

153 mL C2H4O2 per liter, 10 mmol/L TPTZ (2,4,6-tripyridyl-s-triazine) solution in which 40 mmol/L

154 HCl solution was used as the solvent, and 20 mmol/L FeCl3·6H2O. The working FRAP reagent was

155 obtained by mixing 100 mL acetate buffer, 10 mL TPTZ solution, and 10 mL FeCl3·6H2O solution



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156 [32]. One hundred microliters of different amounts of FeSO4 solutions or extracts were added to 6

157 mL FRAP reagent and incubated in a 37ºC water bath for 30 min. The wavelength of 593 nm was

158 used to monitor the absorbance of the samples.

159

160 DPPH assay

161 Sixty micromolar DPPH was dissolved in 3 mL ethanol, and 0.5 mL of different amounts of

162 extracts or OPC were added. Blank experiments, which contained 0.5 mL of 99% ethanol instead of

163 the extract, were also conducted. The absorbance values were monitored at 517 nm [33]. The

164 inhibition rate (IR) of the DPPH free radical was calculated using the following equation [34]:

165 0 0( ) / 100%SIR A A A (3)

166 where A0 was the absorbance of the blank experiments, and As was the absorbance in the presence

167 of the samples. When the IR is 50%, the corresponding concentration is called the inhibition

168 concentration 50 (IC50). Therefore, the value of the IC50 was obtained by fitting the sample

169 concentration and inhibition rate [35].

170

171 Statistical analysis

172 Statistical analysis of the single-factor experimental data was performed using SPSS 11.5

173 software (SPSS Inc., Chicago, IL, USA). Dunnett’s one-tailed t-tests permitted the examination of

174 the statistical significance of the means in the different levels of parameters. Stat-Ease

175 Design-Expert 8.0.0 (Trial version, Stat-Ease Inc., Minneanopolis, MN, USA) was used for the

176 experimental design and regression analysis of the experimental data. Student’s t-test permitted the

177 determination of the statistical significance of the regression coefficient, and Fischer’s F-test

178 determined the second-order model equation at a probability (P) of 0.001, 0.01 or 0.05. The



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179 adequacy of the model was determined by evaluating the lack of fit, the coefficient of determination

180 (R2), and the F-test value obtained from the analysis of variance (ANOVA) that was generated. All

181 the experiments were conducted in triplicate, and the average values ± SD (standard deviation) were

182 calculated.

183

184 Results

185 Single factor test results

186 Effect of extraction voltage in single factor analysis

187 The extraction voltage was one of the important factors affecting the yield of total flavonoids

188 [36]. The effects of different voltages (60, 80, 100, 120, and 140 V) on the yield of total flavonoids

189 were investigated, while the other extraction conditions were as follows: ethanol concentration of

190 70%, ratio of ethanol to raw material of 30:1, extraction time of 60 s, and extraction number of 3.

191 The results showed that the yield increased greatly when the voltage increased from 60 to 140 V,

192 and the highest yield was obtained at 120 V (Fig 1A). With the increase in voltage, the yield of

193 flavonoids increased. This phenomenon might be attributable to the increase of the rotational speed

194 of the head of the flash extractor, the increase in the temperature of the extraction solvent at the

195 same time, and the flavonoids undergoing side reactions, such as oxidation or decomposition,

196 resulting in a decrease in the content of flavonoids [36]. In this experiment, the optimal extraction

197 voltage was considered to be 120 V.

198

199 Fig 1. Column chart showing the effect of different factors on the yield of total flavonoids. The

200 effect of (A) different voltage, (B) ethanol concentration, (C) ratio of ethanol to raw material, (D)

201 time and (E) extraction number on the yield of flavonoids was determined. (t-test, * p < 0.05, ** p <



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202 0.01, *** p < 0.001)

203

204 Effect of ethanol concentration in single factor analysis

205 Different concentrations of ethanol (40, 50, 60, 70, and 80%) were utilized to study the effect

206 of the ethanol concentration on the extraction rate, while the other experimental parameters were as

207 follows: the extraction voltage was 120 V, the ratio of liquid to material was 30, the extraction time

208 was 60 s, and the number of extraction was 3.

209

210 As shown in Fig 1B, the extraction yield of the flavonoids was greatly influenced by the

211 concentration of ethanol [37, 38]. This yield increased with the augmentation of the ethanol

212 concentration until it reached a maximum production of 37.53 ± 1.12 mg/g at 60% of ethanol. It

213 also indicated that the maximum extraction yield was obtained with 60% ethanol. Therefore, 60%

214 ethanol was used as the center point for the additional RSM experiments.

215

216 Effect of the ratio of liquid to material in single factor analysis

217 The effect of the ratio of material to liquid on the yield of flavonoids is also important [39, 40].

218 Different ratios of liquid to material (20, 30, 40, 50 and 60) were designed to investigate their effect

219 on the extraction rate. Simultaneously, the other conditions were as follows: extraction voltage of

220 120 V, ethanol concentration of 60%, extraction time of 60 s, and number of extraction of 3. As

221 displayed in Fig 1C, the yield increased strongly as the ratio increased from 20 to 60. When the

222 ratio of the liquid to the material continued increasing, the output was maintained at a stable level.

223 Therefore, the ratio of liquid to material of 40 was used in this study.

224



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225 Effect of extraction time in single factor analysis

226 Using a fixed extraction voltage (120 V), ethanol concentration (60%), ratio of liquid to

227 material (40) and extraction number (3), the effects of different extraction times (30, 60, 90, 120,

228 and 150 s) on the extraction rate of the flavonoids were determined. The yield of the flavonoids

229 increased significantly with the increase in extraction time from 30 s to 150 s (Fig 1D). However, at

230 more than 90 s, the flavonoid production decreased slightly. This finding may be attributable to the

231 prolonged extraction time; a large amount of heat is generated due to the rapid rotation of the head

232 of the flash extractor; the temperature of the extraction solvent is increased, and the flavonoids

233 undergo side reactions, such as oxidation or decomposition, resulting in a decrease in the content of

234 the flavonoids [41]. Therefore, 90 s was used as the center of the extraction time for RSM.

235

236 Effect of the extraction number in single factor analysis

237 The number of the extraction was also important to extract the active compound [42, 43]. The

238 number of the extraction was related to the extraction efficiency, cost, and flavonoid yield.

239 Respectively, different extraction numbers (1, 2, 3, 4 and 5) were used to extract the flavonoid

240 under the optimal parameters obtained above. As shown in Fig 1E, with the increase in the number

241 of the extraction from 1 to 2, the yield of flavonoids increased significantly. However, when the

242 number of extractions was more than 2, the increase in flavonoid production was not significant.

243 Considering the efficiency of extraction, the number of extraction was set at 2 in this study.

244

245 Model fitting and statistical analysis of the extraction process

246 optimization

247 Response surface methodology optimization is more advantageous than the traditional single



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248 parameter optimization, because it saves time, space and raw material. To optimize the four

249 individual parameters of the Box-Behnken design, the 27 experiments designed were completed.

250 Using multiple regression analyses, the following second order polynomial equations are used to

251 correlate the response variables and test variables:

252 Y=-1486.75792+9.06700*X1+0.74617*X2+19.82517*X3+6.21783*X4+0.021275*X1*X2-0.0508

253 50*X1*X3+5.57500E-003*X1*X4+3.90000E-003*X2*X3+0.041075*X2*X4-6.82500E-003*X3*X4-0.0

254 27050*X12-0.039200*X2

2-0.079338*X32-0.10586*X4

2

255 where X1, X2, X3 and X4 were the encoded values on extraction voltage, ethanol concentration,

256 extraction time and the ratio of liquid to material, respectively.

257

258 To determine whether the model was significant, the significance of each coefficient was

259 determined using the F-test and p-value in Table 2. Smaller p-values indicated higher significances

260 for the corresponding coefficient [44]. In this study, the ANOVA of the quadratic regression model

261 demonstrated that the model was highly significant, as was evident from the F-test with a very low

262 probability value (p < 0.0001). With the determination coefficient (R2 = 0.9755) close to 1, this

263 finding indicated that the actual values had a strong correlation with the predicted values [45]. With

264 Adj. R2 values of 0.9469, it could be concluded that more than 90% of the total flavonoid content

265 could be predicted. Only 5% could not be predicted by the model.

266

267 Table 2. ANOVA table for the response surface quadratic model.

SourceSum of

squares

Degree

of

freedom

Mean

squareF-Value

p-Value

(Prob > F)Significant



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Model 2163.56 14 154.54 34.10 ＜0.0001 ***

X1 302.40 1 302.40 66.73 ＜0.0001 ***

X2 416.54 1 416.54 91.91 ＜0.0001 ***

X3 427.09 1 427.09 94.24 ＜0.0001 ***

X4 86.24 1 86.24 19.03 0.0009 ***

X1X2 18.11 1 18.11 4.00 0.0688

X1X3 103.43 1 103.43 22.82 0.0005 ***

X1X4 1.24 1 1.24 0.27 0.6100

X2X3 0.61 1 0.61 0.13 0.7204

X2X4 67.49 1 67.49 14.89 0.0023 **

X3X4 1.86 1 1.86 0.41 0.5334

X12 39.02 1 39.02 8.61 0.0125 *

X22 81.95 1 81.95 18.08 0.0011 **

X32 335.70 1 335.70 74.08 ＜0.0001 ***

X42 597.70 1 597.70 131.89 ＜0.0001 ***

Residual 54.38 12 4.53

Lack of fit 27.50 10 2.75 0.20 0.9760 Not significant

Pure error 26.89 2 13.44

Cor total 2217.94 26

R2 0.9755

Adj. R2 0.9469

Pred. R2 0.9013



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Adequate

precision18.194

268 (t-test, * p < 0.05, ** p < 0.01, *** p < 0.001)

269

270 The failure of the model was represented by the lack-of-fit shown in Table 2. The lack of fit of

271 the F-value was 0.2000, and the P-value was 0.9670, which illustrated that the pure error was not

272 significantly relative. In summary, the model was appropriate. Simultaneously, the adequate

273 precision of 18.194 indicated that the model discrimination was adequate. Therefore, this study

274 model could be used to navigate the design response surface space.

275

276 The P-values of each model term presented in Table 2 indicated that the total flavonoid yield

277 was significantly affected by four independent variables (X1, X2, X3, X4) and all of the quadratic

278 terms (X12, X2

2, X32, X4

2). In addition, the ethanol concentration and extraction time were the most

279 significant factors on the total flavonoid yield followed by the extraction voltage and the ratio of

280 liquid to material.

281

282 Response surface analysis of extraction process optimization

283 The interaction effects of the factors in response surface experiments were displayed using

284 three-dimensional response surface plots (Fig 2) and two-dimensional contour plots (Fig 3). The

285 former described the sensitivity of the variable change on the response value, and the latter

286 illustrated the important coefficients between the different variables [46, 47]. The impact of the two

287 factors on the response could be displayed at one time by these plots. In these figures, the other two

288 factors remained at level zero.



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289

290 The influences of the extraction voltage and ethanol concentration on the flavonoid yield are

291 shown in Figs 2A and 3A. When the ethanol concentration was less than 68%, the flavonoid yield

292 increased, while when it was higher than 68%, the flavonoid yield decreased, and when the voltage

293 was low, the yield changed more significantly. When the ethanol concentration increased from 50%

294 to 60%, the extraction voltage had little effect. However, when the ethanol concentration increased

295 from 65% to 70%, the voltage had a greater effect on the flavonoid yield.

296

297 Fig 2. Response surface method analysis (3D) of the different interaction factors.

298 Fig 3. Contour plots (2D) of the different interaction factors.

299

300 Figs 2B and 3B showed the reciprocal interactions of the extraction voltage and extraction time

301 on the yield of flavonoids. The mutual interactions were significant. With the increase of extraction

302 voltage, the flavonoid yield clearly decreased, especially when the extraction time was longer. After

303 a period of time, the flavonoid yield increased. However, at more than this range, the output did not

304 increase noticeably. Simultaneously, when the extraction voltage was raised to 116 V, the extraction

305 time had a significant effect on the flavonoid yield.

306

307 The effect of extraction voltage and ratio of liquid to material on the flavonoid yield is shown

308 in Figs 2C and 3C. With the extraction voltage dropping, when the ratio of liquid to material was

309 relatively low, there was little effect on the flavonoid yield. However, when the ratio of liquid to

310 material was relatively high, the decrease in voltage had a greater effect on the flavonoid yield.

311 When the ratio of liquid to material increased from 30 to 42.79, the flavonoid yield increased



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312 significantly to 42.79 and began to decline. Therefore, in order to access the maximum increase, the

313 ratio of liquid to material was more effective at 42.79.

314

315 The influences of ethanol concentration and extraction time on the yield of flavonoids are

316 visible from Figs 2D and 3D. This result showed that the extraction time exhibited a significant

317 effect on the flavonoid yield, while the ethanol concentration had a weaker effect. With the

318 extraction time increasing to 87 s, the flavonoid extraction rate increased and then decreased. When

319 the ethanol concentration increased from 50% to 67.79%, the extraction of the flavonoids increased

320 significantly, while at more than 67.79% it decreased. Therefore, to achieve maximum increase, the

321 ethanol concentration used was 67.79%.

322

323 The effects of the mutual interactions between the ethanol concentration and the ratio of liquid

324 to material were significant (Figs 2E and 3E). With the ethanol concentration increasing from 50%

325 to 67.79%, the flavonoid production increased linearly, but at more than 67.79%, it decreased. This

326 finding was probably attributable to the scope of the solvent; the polarity was appropriate and more

327 suitable to extract flavonoids [48]. When the ratio of liquid to material increased from 30 to 42.79,

328 the flavonoid yield increased at first and then decreased.

329

330 In Figs 2F and 3F, the impact of the interaction of the ratio of liquid to material and extraction

331 time was displayed. An appropriate extraction time was essential for the extraction of flavonoids.

332 When the extraction time increased from 80 to 87 s, the flavonoid yield increased, but when the

333 extraction time was longer than 87 s, the yield decreased. The ratio of liquid to material was lower,

334 and the yield also stayed lower. When the ratio of the liquid to material increased from 30 to 42.79,



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335 the flavonoid yield increased significantly, and after 42.79, the change was very small.

336

337 Validation of the model

338 As shown in Table 3, the optimum extraction conditions were obtained, and the flavonoid yield

339 predicted by the Design Expert Software was 66.54 mg/g. To validate the adequacy of the model

340 equations, a verification experiment was conducted under the optimal conditions (within the

341 experimental range): extraction voltage 116 V, ethanol concentration 68%, extraction time 87 s, the

342 ratio of liquid to material 43 and extraction number 2, respectively. A mean value of 66.40±0.80

343 mg/g (N=3) was obtained from the practical experiments. The average was close to the predicted

344 value of the RSM model. Therefore, this model could be used to extract flavonoids, and the yield of

345 flavonoids was higher than that of other extraction conditions.

346

347 Table 3. Experimental and predicted values of the model under optimal conditions.

Optimum

condition

Extraction yield

(mg/g)

Extraction

voltage (V)

Concentration

of ethanol (%)

Extraction

time (s)

Ratio of liquid

to material

Experimental Predicted

116 67.91 87.00 42.79 66.40±0.80 66.54

348

349 FRAP assay

350 As shown in Fig 4, a linear correlation between the concentration of the FeSO4 solution or

351 extracts and the absorbance was displayed. In addition, the concentration of extracts was expressed

352 as milligrams of Salix babylonica leaves per liter. In addition, the total antioxidant activity of the 1



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353 mg extracts approximately equaled the total antioxidant activity of 0.5–0.75 mmol FeSO4.

354

355 Fig 4. Total antioxidant activity of (A) FeSO4 solutions and (B) extracts.

356

357 DPPH assay

358 DPPH is a stable and well-characterized solid radical source, and it is a traditional and the most

359 popular free radical used to assay free radical scavenging activity [49]. OPC, which has a strong

360 ability to scavenge a free radical, was used as the standard to evaluate the free radical scavenging

361 activity of the extracts. As shown in Fig 5, the inhibition rate (IR) and IC50 were used to

362 characterize the free radical scavenging activity of the extracts. With the concentration increasing,

363 the value of the IR increased. The IC50 value of the extracts from the Salix babylonica leaves was

364 0.8937 mg/L, while the IC50 value of the OPC was 0.0196 mg/L. Therefore, the free radical

365 scavenging activity of the 1 mg extracts was approximately equivalent to that of 0.021 mg OPC.

366

367 Fig 5. Scavenging activity of (A) OPC and (B) extracts on DPPH free radicals.

368

369 Discussion 370 Belonging to Salicaceae and Salix, Salix babylonica is a fast-growing deciduous tree, which is

371 widely cultivated in Asia, Europe and America. In foreign countries, the leaves of these trees are an

372 important source of feed for ruminants, particularly in areas that experience harsh environmental

373 conditions. Plant extracts from Salix babylonica leaves contain saponins and other secondary

374 natural metabolites, which can improve nutrient digestibility and feed utilization [50-53]. In China,

375 its leaves, branches, and floes are all included in the Chinese Dictionary of Traditional Chinese



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376 Medicine as a traditional Chinese medicine. The Compendium of Materia Medica recorded the

377 following: Salix babylonica is the lower grade of this product. This plant is cold in nature and tastes

378 bitter. The plant has the functions of hurricane, diuretic, analgesic, and swelling. According to

379 reports, Salix babylonica is rich in flavonoids [54, 55], which have diverse physiological activities,

380 such as anti-inflammatory, anti-tumor, antioxidant, anti-dementia and anti-obesity [56-64]. It must

381 have widespread utilization in the medicine and food industry in the future.

382

383 Salix babylonica is rich in resources and cultivated throughout China, but its medicinal value

384 has not been properly developed and utilized to date. The optimal extraction procedures of the

385 flavonoids from Salix babylonica leaves with new techniques had not been studied frequently, and

386 most of the extraction methods are limited to conventional extraction methods with low efficiency

387 and obvious shortcomings. Xiaoyan Yi et al. used the reflux extraction method, and the yield of

388 flavonoids was 5.15% [10]. Keyue Liu et al. adopted the microwave extraction method, and the

389 flavone yield was 5.67% [65]. The flash extraction method, also known as the tissue fragmentation

390 extraction method, is an emerging extraction method [66-68]. The flash extraction method is

391 applicable to the extraction of the roots, stems, leaves, flowers, fruits, seeds (except for fine seeds)

392 and whole plants and can be entered into the extraction system only by appropriate treatment. In

393 addition to ether and other volatile solvents, water, methanol, ethanol, acetone, and microemulsion

394 [69] can be directly selected as the solvent according to the desired effective site or chemical

395 composition. The extraction process flow is simple, rapid, energy saving, and environmentally

396 friendly. It can be implemented at room temperature, avoiding heat interference, effectively

397 protecting the heat-sensitive components [22, 24] and has obvious advantages compared with

398 traditional extraction methods. In traditional Chinese Medicine research, the flash extraction



https://doi.org/10.1101/423095


399 method will inevitably exert greater potential. In this study, the flesh extraction method was used to

400 extract flavonoids from Salix babylonica. The extraction rate of the flavonoids was 60.4 mg/g

401 (6.04%), which is significantly higher than those of the other extraction methods.

402

403 In the human body, free radicals and oxidants are produced from either normal cell metabolism

404 or external sources. Excessive free radicals and oxidants that accumulate in the human body could

405 generate a phenomenon called oxidative stress and result in oxidative damage to cause the

406 deterioration of the normal function of proteins, nucleic acids and many other biomasses [70]. The

407 antioxidant activity of the flavonoid extract of Salix babylonica leaf was examined by Yaoyao Su et

408 al. who used the ultrasound and microwave methods to extract α-glucosidase inhibitors from

409 weeping willow leaves and found that the inhibition rate of α-glucosidase was 77.85% [9]. Ruiping

410 Ji et al. used indirect iodine quantitative methods to determine the peroxidation of animal and plant

411 oils and fats and showed that the highest antioxidant value of Salix babylonica leaf was 0.204% [8].

412 In addition, Jie Xie et al. used a homogenate extraction technique to extract bamboo leaf flavonoids.

413 The yield of total flavonoids extracted from the bamboo leaves was 8.50 mg/g, which was 20.2%

414 higher than that of the ethanol reflux extraction. The IC50 of the total flavonoids obtained by flash

415 extraction was 67.35 μg/mL, which scavenged free radicals. The effect is better than ethanol

416 refluxing [71]. However, there was no report on the activity of the total flavonoids scavenging free

417 radicals in Salix babylonica leaves. For the first time, this study describes the application of the

418 flash extraction and response surface optimization methods. On the basis of improving the yield of

419 flavonoids, the free radical scavenging activity was maintained at a high level with an IC50 of

420 0.8937 mg/L.

421



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422 In this study, response surface methodology was used to establish the optimal conditions for

423 the extraction of total flavonoids from Salix babylonica leaves using flash extraction. The yield of

424 the flavonoids was maximized and increased significantly compared with those of the conventional

425 extraction methods. In addition, antioxidant assays showed that the extracts with the established

426 procedures in this experiment had stronger antioxidant ability, and 1 mg extracts equaled

427 approximately 0.5–0.75 mmol FeSO4 or 0.021 mg OPC, respectively. These results will help to

428 better exploit the resources of Salix babylonica leaves and provide new insights for the effective

429 extraction of flavonoids with greater bioactivity.

430

431 Acknowledgments432 This research was supported by the National Natural Science Foundation of China (31172295 and

433 31272569). Simultaneously, this research was supported by the University Students’ Scientific and

434 Technological Innovation Research Projects (2014088) operated by the Jilin Agricultural Science

435 and Technology Institute of Jilin Province.

436

437 Authors’ Contributions 438 Conceptualization: Yue Zhang, Guangxing Li.

439 Formal analysis: Yue Zhang.

440 Funding acquisition: Huijie Chen, Lei Diao, Haixin Liu, Guangxing Li.

441 Investigation: Huijie Chen, Lei Diao, Haixin Liu, Ming Zhong.

442 Supervision: Guangxing Li.

443 Writing – original draft: Huijie Chen.

444 Writing – review and editing: Huijie Chen, Lei Diao, Guangxing Li.



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Documents

Optimization of the flash extraction of flavonoids from ...42 Introduction 43 Currently, there are increasing applications of flavonoids in food and medicine for their 44 extensive